MEDICAL ＆ BIOLOGICAL LABORATORIES CO., LTD. 
          5-3 Sakae 4 chome, Naka-ku, Nagoya 460-0008, Japan 
          TEL: +81-52-238-1901 FAX: +81-52-238-1440 E-mail: info@mbl.co.jp 
          www.mbl.co.jp 
               Protocol for Northern Blotting 
               Northern  blotting  was  performed  using  DIG  Wash  and  Block  Buffer  Set  (Sigma-Aldrich;  code  no.  11585762001).  For  more 
               information, please contact Sigma-Aldrich Co, LLC. 
                
               Day 1 
               Electrophoresis and Transfer 
               ↓  Dilute  total  RNA  samples  with  2×Loading  Buffer  (50%  formamide,  6.14% 
                  formaldehyde, 1×MOPS, 10% Glycerol, 0.05% Bromophenol Blue).   
               ↓  Heat total RNA samples at 65°C for 10 min., then quench at 4°C for 5 min. 
               ↓  Load the samples in a 1% denaturing agarose gel (1% Agarose S, 1×MOPS, 2% 
                  formaldehyde), and conduct electrophoresis in 1×MOPS at 50 V for 2 hr. 
               ↓  Rinse the gel in 20×SSC for 10 min, put the following things in order ; one dried 
                  sponge, one filter paper, an agarose gel (upside down), one membrane, two filter 
                  papers, two dried sponges. 
               ↓  Soak the lower sponge in 20×SSC, incubate them at room temperature overnight. 
                  (Please see below.) 
                
                
                
                
                 
                
                                                Sponge 
                                                Sponge 
                
                                                                            Filter paper 
                   Membrane 
                                             Gel (upside down) 
                
                
                                                Sponge 
                                                20×SSC 
                
                
                
                   Day 2 
                   UV cross-link and Hybridization 
                   ↓   Soak the membrane in 2×SSC and put it on the filter paper soaked with 2×SSC. 
                   ↓   Make UV cross-link (120 mJ/cm2) using FUNA-UV-LINKER FS-800 (Funakoshi).   
                   ↓   Put the membrane and the prewarmed PerfectHyb (TOYOBO; code no. HYB-101) 
                       in a hybridization bag at 68°C for at least 30 min. 
                   ↓   Denature the RNA probe at 98°C for 5 min, then quench at 4°C for 5 min. 
                   ↓   Mix the probe solution with 68°C prewarmed PerfectHyb. 
                   ↓   Discard the solution and put the probe solution in the hybridization bag, incubate 
                       the  membrane  at  68°C  for  16  hr.  (The  condition  of  the  hybridization  reaction 
                       depends on the probe being used.) 
                    
                   Day 3 
                   Wash step after hybridization 
                   ↓   Wash the membrane twice with Low stringency buffer (2×SSC, 0.1% SDS) at room 
                       temperature for 5 min. each. 
                   ↓   Wash the membrane twice with High stringency buffer (2×SSC, 0.1% SDS) at 68°C 
                       for 15 min. each. 
                   ↓   Rinse the membrane with Wash Buffer for 2 min. 
                   Detection 
                   ↓   To reduce nonspecific binding, soak the membrane in Blocking Buffer at room 
                       temperature for 30 min. 
                   ↓   Incubate the membrane with Anti-Digoxigenin (DIG) mAb (MBL; code no. 
                       M227-3) diluted with Blocking Buffer at room temperature for 1 hr. 
                   ↓   Wash the membrane twice with Wash Buffer for 15 min. each. 
                   ↓   Incubate the membrane with 1: 5,000 of Anti-IgG (Mouse) pAb-HRP (MBL; code 
                       no. 330) diluted with Blocking Buffer at room temperature for 1 hr. 
                   ↓   Wash the membrane twice with Wash Buffer for 15 min. each. 
                   ↓   Wipe excess buffer on the membrane, then incubate it with appropriate 
                       chemiluminescence reagent for 1 min. Remove extra reagent from the membrane 
                       by dabbing with paper towel, and seal it in plastic wrap. 
                   ↓   Expose for 1 min. with ImageQuant LAS 4000 imaging system (Fujifilm). The 
                       condition for exposure and development may vary.